Biochimie {1998) 80, 37 ! -377
O Soci6t6 fi'an~;aisede biochimie et bi~lo,,ie molOcukfire t Elsevier, Paris
Physica| techniques for the study of exocytosis in isolated cells
J P H e n r y ~', F D a r c h e n , L S C r i b i e r b
Neurobmiogte Physico-Chimique. CNRS ERS 575:
t hvswo-Chmm' Mol&'uhtire des Membranes Bio/ogiques, CNRS-UPR 9052, h~stitut de Biologie Physico-Chimique,
13, rue Pierre-et-Marie-Curie, 75005 Paris. Fram'e
l)
j
•
•
•
(Received 8 December 1997: accepted 16 March 1998)
S~mmary ~- Membrane traffic is an fluportant aspect of cell biology which implies shuttle vesicles and multiple binding/fusion event,;. In
spite ot' rapid progress at the biochemical level, the mechanism of fusion is still not understood. A detailed physical description of the
phenomenon in possible at the level of the plasma membrane where secretory ,,esicles fuse with the cell membrane, a process known as
exocytosis. Tiffs process is specially active in neuron~ (release of neurotransmiuer) and in endocrine cells {release of hormones). ~here
exocytosis is tightly regulated, Among the biophysical techniques developed, cell membrane capacitance measurernents by the technique of
patch-clamp and amperometry of the oxidizable secretory produc|s have resulted m interesting intormation. These techniques llave described
the initial fusion pore, its fluctuations, the efflux of material through the pore and its irreversible expansion. Optical teci'|niques, using
bioluminescent and fluorescent probes are also in progress. For instance, the dye FM 1-43 binds to but is not ~ranslocated through biological
membranes and it has been used to measure membrane surface, as done by capacitance measurement. Evanescent wave fluorescence microscopy has been recently introduced to analyse the behaviour of secretory granules ill the vicinity of the plasma membrane (Q Societ6 franqaise
de biochimie el biologic inol6cuhlire / Elsevier, Paris).
membrane htsion I exocytosis I patch-clamp / amperometry / evanescent wave microscopy
M e m b r a n e fusion is an i m p o r t a n t biological
phenomenon
Cells are limited by a i~hospholipidic bilaycr, the plasma
nlenlbraue, but they contain much more membrane surface
inside than around then!, These internal membranes dcl'inc
specialised compartments (nucleus. inilochondria, cntioplasmic reliculum, Golgi apparatus, secretory vesicles, cndosomes, peroxisomes, etc) where differetlt chemical
reactions can be perlbrmed at the same time under optimal
physico-chemical conditions. Several of these comparto
ments communicate with each other. This is the case for the
compartments composing the secretory pathway (endoplasmic reticulum, Golgi apparatus, trans-Golgi network and
secretory vesicles) and the reverse endocytotic pathway (endosomes, lysosomes). It is now clear that the main stream
of the corresponding traffic involves shuttle vesicles, budding from the donor compartment and fusing with the accepter compartment. This traffic occurs in all cells and it is
absolutely nece,;sary Ik~r them. In tiffs respect, membrane
fusion appears to be an universal and important phenomenon. At the present time, our knowledge of tht ° proteins
Abbreviations: HA protein, inl'luenza Ilemagglutinin; 5-HT. sere-
tents: AFM, atomic three microscope; GFP, green fluorescent protein, CALl, chromophore-assisted laser inactivation.
involved in this process is progressing rapidly, though tile
mechanisn! of fusion in still not understood 11~1.
The relation of cells with their environment implies also
various types of membrane fusion. For instance, many
viruses arc enveloped by a phospholipidic bilayer and their'
entry in the cytoplasm requires membrane fusion° The be~t
known model is Itle illl]llCllZa virus, which i~ first taken tip
by cndocytotic vesicle%, Fushm occurs betwcea tile virw,
and the endocytotic vesicle and is triggered by the acidifio
cation of the vesicle. Only one viral protein is required for
fllsion, influenza hemagglutinin (HA protein) and the pro°
cess of fusion is the subject of many investigations. How°
ever, in this review, we would like to f,acu~ on exocytosi~
associated with regulated secretion. Hydrophilic hormones
and n e u r o l r a n s m i t t e r s are a c c u m u l a t e d in secretory
vesicles, and upon stimulation, the secretory cells release
these products by fusion of the secretory vesicle membrane
with the plasma membrane. This process, named exocyo
tests, is characterised morphologically by ~2 images, in
which the vesicle is open to the external milieu.
Exocytosis is a rapid biological phenomenon. In the
nervous system, neurotran.,;mitter release, detected by incase
urcment of the electrical response of the posto~ynaptic cell.
occurs within 100 Its after action potential arrival, in endocrine cells, exocytosis is less rapid, though secretion is
generally induced by the same second messengc|; an in~
crease of intracelluhtr calcium ion concentralion. Exocy~
tests of the catecholamines adrenaline and noradrcnaline
372
maining part of the cell melnbrane at a given potential gives
the activity of the ionic channels present in the cell.
If, under the same setting, the capacitance is measured in
place of the conductance, the technique will allow determination of cell area 151. Exocytosis will increase this area by
adding the surface of fused secretory vesicles whereas endocytosis, the reverse phenomenon, will decrease the cell
surface. In this technique, it is assumed that the conductance
increase is only related to the surface increase and that there
is no variation of the dielectric factor, due tbr instance to
the assembled fusion pore. This technique was first applied
to chromaffin cells, the secretion of which was stimulated
by raising the intraceilular Ca 2+ concentration through the
pipette or by depolarising the cells. A large increase in membrane capacitance was obselved which was lbllowcd by a
slower decrease. With the whole cell approach, unitary steps
corresponding to the fusion of a single secretory granule
contained in a chromaffin granule, the secretory vesicles of
the adrenal medulla chromaMn cells, requires about 20 ms.
These ~ l l s can be obtained quite easily in primary cultures
and they have been developed as a good model to study
laffin granules (270 nm
vesicles {50 nm diameter)
Electrical m e t h ~ d e v e l o ~ to describe exocytosis
In the technique of patch-damp, a cell is immobilised at the
tip of a micropipette, under conditions where the electrical
leak between the cell membrane and the glass is low (gigaohm
resistance seal), in the whole cell c~mfiguration (fig I, upper
part), the membrane patch inside of~he pipette is sucked off
and measurement of the current flowing through the re-
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I'11!I, EIt~tricalapprt~he.~ us~ to 11~easureext~ytosis. Upl~r lel'~,patch-clamp iu the svhule-cdl configuration. This technique is used to measure
the cell ~udh~.~increase, 11~asurtMas a zat~tcitant~ increase, resulliug from the Ihsion of the Su~l"eloIT~esicle. in the ini|ial s|ep. a IX)J~is fi)nned
which has a n~asurabl¢ cotldu~tatlce g. Oxidizable sec~R.'xlI~xlucts are delecttM t~y ami~rt~metl~,,using a cal"hm fihl~ set at a ix)sitive potential.
UPl:~rdght, ceil Cal~citant.~antt am~wmetry n~asur~Msimultantx~uslyon a beige mouse mast ceU.The I~re fluctuatesl~l'orc irreversibleexpansion
arm rdea.~ of 5 - ~ (taken fi'om IZOI). Lower left, th~ t~solutiol~of the technique is improvtxl when patch~lamp is peit'om)ed in the cell attach~M
configuratitm. The aml~rometric c~u~n fibr~ is intrtxluced in the glass mictopilx.tte. Lower right, simultaneous measurements o|" ampc~x~mctric
spikes {la). capacitant'~ (C) a~M~o~Muctanceat" the thsion I~*~ {G) on a chwmatli, cell (taken |'~x~m181).
373
could not be resolved. However, this can be done on other
secretory cells, for instance, the mast cells of a mouse mutant, the beige mouse, whicll contain few ~1-10) bu| ku~2er
(2-6 l.tm diameter) secretory granules (fig I. upper right
part). These cells are involved in inflammation and. in the
presence of immunoglobulins of the lgE class, they secrete
Mstamine and serotonin (5-hydroxytryptamine). With these
cells, Zimmerberg and colleagues have observed unitary capacitance jumps, ranging from 0.1 to 0.8 pF [6]. They could
correlate the capacitance jump with direct observation of
exocytosis, using differential interference optics. Interestingly enough, they observed that the granules swelled (see
below), but alter the capacitance jump, thus indicating that
the swelling cannot be the initial driving force for membrane fusion. Recently, the sensitivity of the technique has
been increased by shifting the patch-clamp configuration
from whole-cell recording to the cell-attached one Ifig 1,
lower part), in the latter case, the isolated membrane patch
is that present in the pipette (which is not sucked off). Because the naeasurement is restricted to a small area. the signal-to-noise ratio is increased 15, 7, 8[. This has been
applied to the study of chromaffin cells, where unitary
jumps were estimated to be !-2.7 fF 15, 8, 91, figures which
are consistent with the size of a chmn:affin granule (270
nm) and a membrane capacitance of I laF/cm-'.
Another i n t e r e s t i n g d e v e l o p m e n t of the electrical
methods is the measurement of the conduclance of the initia! fusion polv. Two approaches have been described. 1,1
the first one 161, according to the equivalent circuit describing the adnltttanc
' ' ' "e of a ccl
"" I to which a sine wave is applied,
the voltage drop that occurs during exocyto,;is is considered
to include an ohmic component resulling from the resistalice of the fusion p( ic, initially very small (fig I, upl;~er
part). In t!~e second approach, advanlagc is taken l'ronl Iil0
l~lct thai secretory granules arc generally pohuised I I0l. An
ATP-dcpcndent clectrogenic I I~-pumlx present in tile tnembrane of secretory granules, generates a transmembrane
potential wilh the granule matrix positively charged with
respect to the cytosol, During fusion, the efflux of these
charges in the external medium generates a transient current, fi'om which the conductance of the port: can be derived.
These two methods were originally devised on mast cells
from beige mice and they gave convergent results, with an
initial pore having a conductance in the range 250-350 pS.
Such a pore would have a diameter of !-2 nm, smaller than
those observed by electron microscopy. Solving the admittance equation gave indications on the evolution of the l'ustun pore. Generally, the diameter of the pore increases
extremely rapidly in the first 10 ms and then more slowly
for about 150 .us. in some instances, the pore flickered (fig
I, upper part) and finally closed, thus indicating that fusion
is initially reversible and does not lead necessarily to complete exocytosis.
More recently, this approach has been applied to smaller
secretory vesicles, such as the chromaffin granules of chromaffin cells 18 I. in this case, the cell attached configuration
,
,} ,., ,,
~fi,,~ l, lo,aer pari~ and a higher frequency sine v,a,,e have
been used iTD, The results are basically similar. ~ith a d i s
lribu|ion of the conductance of lhe initial fusion poxv ce~,red at .'~60 pS (correspondim,e. to a diameter of 1.7 nmk rapid
enlargement of the pore or. more rarely fluctuations of the
pore without complete fusion.
To summarise the inlbrmation obtained by these techniques, exocytosis, which can be monitored by capacitance
measurements, gives rise to a fusion pore. with a welldefined conductance. The size of this conductance is in the
range of that of large ionic channels and thi:; observation
has suggested that fusion, in its initial state, might involve
a proteic structure, on one membrane or on both. However.
this issue is largely controversial, and others have suggested
that the pore might be purely lipidic, a hypothesis based on
the size heterogeneity of the fusion pore and on its "slow"
kinetics[I, 31.
A m p e r o m e t r i e m e t h o d s u s e d to m e a s u r e the release o f
the secretory products
Tile catecholamines adrenaline, noradrenaline and dopaminc, which are common secretory products of neuronal
and endocrine (chromaffin) cells can easily be oxidised to
quinones at the level of a carbon electrode set at a fixed
positive potential. Oxidation generates a current and this
technique is an exquisitely sensitive assay of the sccrctkm
products ! I i, 121 The current is proportional to the conceno
,ration of catecholamine and it monitors directly the rate of
release. !tl the usual setting (fig !, upper part), a carbon
microl°ibre is placed adjaccn! to a catcclmhmlinergic cell,
such as a chromaffin cell. WI!en the cell is stimulated by
various illcans increasing the intracellular Ca ~'* concemrao
lion, an oxidation current is observed, which is attributed to
the release o f cateclmhuninc,,. The sensitivity and the time
resolution of the techt~:, ,~ arc .,,ueh that individual pc;~k~
corresponding to the rcizuse of one secretory granule can
easily be resolved (fig !). Quantitatively. integration with
time of the spike current gave figures of about I pC° correo
spending to about 3 x 10¢,catecholamine molecules in a throe
malTin vesicle. Theses figures are consistent with the estimated
molar concentration of vesicular catecholamines (0.5 M) and
the size of the vesicle (270 rim). Practically, such peaks have
a height of several tens of pA and a duration of about 20 ms,
depending upon the spatial relatiortship of the cell and of the
electrode I! 2, 131. Ill fact. this setting has been compared to a
synapse, the electrode working as a post°synaptic receptor
generaling a current in response to the release of the neumo
transmitter. The same technique can be applied to other dec°
t m o x i d i z a b l e secretory products such as serotonin
(5-hydroxytryptamine, 5-HT) or cvcn insulin J!41.
The amperometric approach has opened many possibilities. An interesting one is the theoretical analysis of the
shape of the amperometric spike 115, 161. if the electrode
is at short distance from the cell. this shape will reflect 111e
374
rate of catecholamine release. Various factors have been
considered as being rate-limiting in the kinetics of release:
the irreversible expansion of the pore and the diffusivity of
the secretory molecules in the granule matrix. It has to be
realised that in the matrix, monoamines are cations which
;the
pro~iast
histamine ate present in the matrix as divalent cations at the
intragranulat pH, During exocytosis, counterion replacement will be done by cation exchange with Na + ions present
in lhe medium. Displacement of divalent monoamine cations by monovalent Na* induces a swelling of the gel by
an osmotic effect. This phenomenon has been studied by
atomic force microscopy (AFM) 1191. Therefore, the matrix
can have several effects on the release of monoamines: i)
the swelling of the gel may be the driving force controlling
the irreversible expansion of the pore; it) the diffusivity of
the monoamines may be regulated by an ion exchange process at the level of the gel; and iii) the diffusivity of the
monoamines may be controlled by the swelling of the gel.
The two first effects have been analysed theoretically and
are supported by experimental data.
Very often, the amperometric spike previously described
is preceded by a small 'foot' or 'pedestal' ! 131. This phenomenon has best been investigated by coupling amperemetric and capacitance measurements. The analysis, first
performed on the release of giant vesicles from beige mouse
mast cell."s I I 2.201. indicated that the foot is associated with
transient vesicle fusion. Serotonin is released through the
filsion pore, belbre the irreversible expansion which leads
to the amperometric spike, During the Ibm period, the rate
of release estimated h'om the mugnilude o1' the amperomeo
tric current is proportional to tile size of the pore. derived
ITem the conductance. Wllen tile pore is fluctuating, the
ampemmetric signal follows the same kinetics.
The repetition of this type of experiment on the exocytests of the smaller secretory granules of chromaffin cells
has b~n made possible by improvement of cell capacitance
measurements 181, In this case, as described p~viously, cell
~:apacltance is observed in the cell attached configuration,
on the membrane patch electrically isolated inside of the
pipette (fig I, lower part), Amperometric measurements
thus ~quim the carbon micmfibre electrode to ~ placed
inside the patch-clamp pipette. Again, n:lease of the secretory product, catecholamin¢, was observed through the
thsion pore, before irreversible expansion, Catecholamine
release was also observed during pore fluctuations without
a rapid amperometric spike (more than 10% of the ext~yretie events). These 'stand alone' Ibm signals are thm~ght to
r~fleet transient I'usions,
Interestingly enough, estimation of the amperometric
signal indicates that virtually all the monoamine content of
the vesicle can be released during such transient fusions,
This result differs from that observed in the giant vesicles
of mast cells 1201, which release only a few percent of their
content during transient fusion. The difference can be explained in t,,,o ways: i) though there is a large volume difference between the two kinds of secretory vesicles, the size
of the fusion pores is comparable; and it) in mast cells, the
diffusivity of the amines seems to be more controlled by the
influx of the counterion than in chromaffin cells ! 17].
The observation that in chromaffin cells, exocytosis
could occur without irreversible expansion of the pore is
interesting from the theoretical point of view, since it lends
some support to the 'kiss-and-run' hypothesis 1211, According to this hypothesis, first proposed for neurons, the last
release of neurotransmitter would not involve complete fusion. the neurotransmitter diffusing rapidly through a transient pore. Such a kiss-and-run mode would be more
adapted to release by neurons than by endocrine cells Ibr
several reasons: i) a higher time resolution is required in the
case of neumtransmission where trains of impulses are
often seen; it) synaptic vesicles (50 nm diameter) are much
smaller than chromaffin granules (270 nm diameter) and
diffusion through a 100-500 pSl i nm diameter pore would
mobilise the neurotransmitter extremely rapidly (less than
I ms); iii) such a pore would not be permeant to matrix
polymers, but synaptic vesicles do not contain any releasable soluble protein. If, as discussed above, the initial pore
were of proteic nature, the kiss-and-run hypothesis would
suggest that exocytosis occurs without phospholipid membrane fitsion, a provocative hypothesis.
Ill stnllnlary, amperonletric nleaSUl'enlellts confirm and
precise the biphasic nature of exocytosis in endocrine cells.
They suggest that the polymers present in the matrix of
secretory granules from these cells play an i!u!~or!an! role
in the expansion of t!!e pore and the release ol' hormones,
They ~tIl~pOrllhe k is~oaud run hYl)olhcsis, a mode of ivlcase
which might be itnportant i11 neuml~s, where ~ynaptic
vesicles do not conlaill p~dymer inalrix.
Optical t~hniques to study cellular traffic and
regulated exocytosis
Association of exocytosis with a light emitting reaction
In parallel to dectrical and electrochemical tedlniques, eflbrts have been made to develop optical techniques to visualise vesicles undergoing fusion. For instance, it is
possible to associate a light-emitting reaction with the release of the secretory products. Chromaffin granules conlain high c(mcentn~tions of ATP (0,15=0,25 M) co-t~leased
with catecholamines that can be assayed by a bioluminescenc¢ reaction, using tile firefly enzymatic system 1221.
Light emission by these insects results from the oxidation
of a substrate, the firefly luciferin, by its cognate enzyme,
lucil~rase, in addition to O2, this reaction requires ATP and
the intensity of the light emitted is proportional to ATP concentration. The sensitivity of the assay is high, reflecting a
375
high quantum yield. Thus, if chromaffin cells arc stimulated
in a medium containing firefly luci|'erin and luci|~rase, reab
time measurements of secretion call be performed. As |'~r
amperometric measurements, the intensity of the emh~ed
light is proportional to the rote of emission.
Recently, the same idea has been followed, but using a
more sophisticated system in which the secretory cells have
been genetically engineered [231. Another bioluminescence
enzyme, the luciferase of the crustacean Cypridina, was expressed in neurons and t~rgeted to synapfic vesicles, This
has been obtained by constructing a chimera between the
iuciferase and a vesicle membrane protein (named synaptolucin), in which the luciferease part was expressed in the
vesicle lumen. Cvpridina luciferase emits light in the
presence oI' tile corresponding iuciferin (which is different
i'rom the firefly luciferin) and of O2, This molecule, luciferin, is a relatively impermeant guanidhle derivative and,
when added to a culture of engineered hippocampal neurons, no light emission is observed. However, during cxocytosis, synaptolucin will have access to the external
medium and will give rise to light emission. If some luciferin molecules are internalised in the vesicles during the
endocytotic step which follows the release, it will be rapidly
consumed. Therefore, light emission should monitor the secretory event. At the present time, the sensitivity of the technique does not allow measurements at the level of a single
synaptic vesicle (which is much smaller than a secretory
granule), but this approach might be promising.
Activity-depemh, nt,lh,Jrescem staining amt &,staining o.f
secretory cells
This approach, which has been developed in the htboratory
of Betz is based upon the use of a fluorescent styrene dye,
FM 143, with original characteristics. TI!is an lphil)hilic
compound has a fluorescence wllich is low in aqtleous st~lutions and high when bound to memblane,. However, it
bears two positive charges which limit ils translocation
through the bilayer and it stays on the cis monolayer (on the
monolayer facing the medium where the dye has been
added). Thus, the amount of membrane°bound, fluorescent,
FM 1-43 monitors the membrane area in contact with the
external medium, thus giving information similar to that
obtained by capacitance measurements.
The similarities and the differences between two approaches have been determined on chromaffin cells 1241.
Incubation of the dye with resting cells stains the plasma
membrane in a stable manner; a brief stimulation of the cells
in the presence of the dye increases the fluorescence of the
cell and this increase is proportional to the capacitance increase. In the proposed experimental approach, no el'l'ort
was made toward the elementary fusion event, probably because of technical diMculties. However, an important finding was that the fluorescence increase, corrected for
photobleaching, had a higher stability than the capacitance
increase, on a time scale of 30 s. Capacitance measures the
nel increase of lhe plasma membrane surface, and it is a halo
amce bet~veen exo- and end~ytosis. On the other hand. the
stained membrane which is intemalised w{fl stay fluorescent
and the fluorescence of the dye ~vilJ thus integrate the whole
history of exocytoti~: events. A consequence is that staining
with FM 1-43 can be used to folkway the late of the secretory,
granule membrane after exocytosis, though no imaging ex~riment has been performed in chromaffin cells.
In fact, the FM 1-43 dye has mainly be used on neuronal
preparations, such as the frog neuromuscular junction, to
monitor the full exocytosis-endocytosis sequence [25]. At
variance with endocrine cells, neurons utilise several times
their secretory vesicles (synaptic vesicles). After neurotransmitter release, the synaptic vesicle membrane is retrieved by endocytosis, the ,,~".cslcle is filled again with
neurotransmitter by a vesicular uptake system and then it
can engage in a new exocytosis round. When the frog nervemuscle preparation is stimulated in the presence of FM 143, the nerve terminals are stained. A fraction of the staining
is rcsl,"
"s't,mt to washing, corresponding to the dye internalised within synaptic vcsiclc
- " - ~s
.... This contention is supported by the fact that a new stimulation of the preparation
in a dye-free medium destains the nerve terminal [126!.This
lechnique gives interesting insight into the secretory cycle,
for instance, using this approach, it was concluded that the
full exocytosis-endocytosis cycle in this preparation requires a total time of 1 rain [27]. However, at the level of
the fusion events, the dye does not seem to be a simple
marker of exocytosis 1281.
In its prese,lt state, the approach has not been developed
for imaging purposes. Under the fluorescence microscope,
|he labelled terminals are c mactcnscd by fluorescent spots
which are interpreted as synaptic ,.c:s!t:! s clusters, At the
ullrastructurai level, hislochemical techniques show that the
dye is prominently associated with vesicles [2(~1, but these
small s'I,,.c, vesicles are packed in clusters and the individual
vesicles cannot be re~olved by ftuot~e~¢ence microscopy.
Imaging exocytosis by evanescent wave,flllores¢:ettc¢
Ill
iI~,I~iSCI)py
Imaging individual lilsion events is impeded not only by the
size of the secretory vesicles, but also by their number, in
chromaffin cells, with a diameter of about 20 ~m, there are
10 to 30 000 secretory granules with a mean diameter of
270 nm. It is possible to label the granules with fluorescent
markers without affecting the secretol'y capacity of the cells.
In this case, the FM 1-43 dye is not suitable since the Seo
cretory granules ate not recycled directly after endoeytosis,
but acridine orange is a fluorescent permeant weak amine
which is taken up by the granules 1291, as a result of the
ApH existing between the cytosol and the acidic granule
matrix (two pH units, giving rise to a two-order of magnitude concentration gradient). Alternatively, chromaffin cell
lines have been engineered genelically to express in the
granule matrix the green fluorescent protein (GFP) of the
~6
jelly fish Aequorea 1301. Of the thousands of granules present in a cell. those undergoing exocytosis after a brief stimulation are located at short distance from the plasma
membrane. In fact. ultrastructural studies [291 showed the
existence of a ptx~l ¢ . . . . .
, in contact with the
2000 granules/cell,
and analysis of this
~tive.
le of evanescent-wave fluorescence
microscopy has been introduced to examine the fluorescent
granules pre~nt in the vicinity of the cell membrane [29.
301. An evanescent wave is generated at a glass/aqueous
medium interface by a laser beam suffering total reflection.
Chromaffin cells, cultured on the glass interface, are excited
by the evanescent wave and. since the energy of the evanescent wave decays exponentially along the Z axis, only the
granules present in the 300-nm thick proximal layer will be
excited. Because this depth is comparable to the diameter
of the secretory granules, the technique is perfectly suited
for the study of docking and of subsequent steps, including
fusion which is visualised as a sudden vanishing of the fluorescence signal, as a result of the diffusion of the secretory
products. The technique is promising, for instance ibr testing the existence of specific release sites or for investigating
factors controlling the movement of organelles before and
after fusion.
Conclusions and perspectives
We have presented the most informative approaches dee
r e l o a d in recent years. In this rapid presentation, we have
not st~ssed systematically the limitations of the various
techniques. For instance, with the i~atcla-clan~p metlaodoo
Iogy in the whole-cell configuration, the cell content is in
with the buffer p~sent in the pipette, thus
c omrol of the internal medium, but it is
difficult m change rapidly the composition of the pipette
medium during the course of an experiment, For Ca~* and
various nucleotides, this has been done by using caged
molecules 131, 32], in which the reactive species is combined with a photosensitive dye which masks its activity,
Illumination of the caged compound releases very rapidly
the desired slxx~ies at a concentration which can be modulated by the intensity of the flash,
In the case of the patch-clamp in the cell attached configuration 181, the stimulation of the secretion is not controlled: most of the cells could not be stimulated by
electrical depolarisation and some responded spontaneously
during seal t'ormation,
The ampemmetric approach is very attractive because it
is non-invasive, the electrode being out of the cell, However, this in turn raises st~me problems since the response is
dependent upon the geometry of the cell/eleetrt~le pair:
in spite of these and other difficulties, many inte~sting
results have Ix~n harvested by these techniques, We have
selected data more directly involved in the fusion process,
but other data have been obtained, tbr instance, on the size
of the readily releasable pool of vesicles, obtained by the
capacitance technique [33, 34], which depends upon some
prefusion steps.
What is the future of these techniques'? The propagation
of the action potential associated with nerve conduction has
been formally described long before its molecular basis was
understood. A similar quantitative description of exocytests, including the various prefusion steps, similar to that
performed by Katz and his colleagues on nerve impulse
propagation, is under its way in several laboratories, including that of E Neher. Another line o1" research of this laboratory is to apply the same techniques not to isolated
chromaffin cells, but to tissue slices, to avoid ~zrlefacts
caused by cell culture. This has recently been done on adrenal slices t'rom mouse 1351. These experiments ate interesting tk~r two reasons: i) in slices, the cells keep their
environment and their polarity, wMch is lost in isolated
cells; and it) mice can be manipulated genetically, thus
allowing the study of different genes involved in the secretory process through analysis of the exocytotic response.
Biochemical and genetic approaches point to various
proteins which might be involved in the secretory process.
The function of these candidates has to be validated by the
biophysical approach. This can be done in many different
ways: cells isolated from genetically modified animals, expression or microinjection el' recombinant mutant p~x~teins,
microinjection of antibodies or peptides. It might also be
possible to use chromophore,assisted laser inactivation
(CALl), which causes the precisely tuned thermal denaturation of specific proteins by laser light targeted through a
dyeolabe!led antibody !361. Identification ot' Ihe al'l~cted
step give~ some it)dication oft the l'tulction o1' a protein,
More specifically, a breitkthrough would be the identifica,
ties of the protein(s) involved in the f~)rntation of the l'usion
pore, either lipidic or pmteic.
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15, '~
"~ '
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~1
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,afc..lh:iunl-dcliCnd...:nlcxo..'3ira,i,,illpittimff) nlclartc~Ir~ph'..C.mllpari>,~ilm ihc pll~mlhlbih: cah. mm chclai~r~ mti~q~hcm. ~.I (;'IL%and I)M
niirol~hen. ('vll {'.h'i.m Itl, Ib15-.-I~2
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,_,ranulc,.. lht: u.x.tlt.'}l,.ili...
bur,.l, and illc nccd Ior ,.%1"i~'h~dr....l),,l.,eli
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t4 Iiilli,, K I ) , M l l ~ l l C r R, Nt, h¢l < l; I[IJtJf]i lth!lClll kiiia~c ( tllilanc:c~
c%lw%lll,~h IFil!ll t Jllltill'.ilIill CL']]'~ tl% Ilitrtv.t~liiQ Ihc ~i/c t!l lhc ICddlJ%
ivIca~illiiC Ihiill I~l ~ctit!h!i)~lalilit~,'~, ,%'cu~i,. il:t, l.l.{)tJ I.].?ii
lti Mil~,i>il' +l< Nciilir I: ( ]lJqlTi Rillnd t'%iit'~, liiM~, eli .~tllTJc ehi~illhtflili ccll~
iC~)II!IICll Illllll Illitll'~C ,litll,'ltill ~ Itt"~ ] ,~,'l'illf~i # 17 ~ { t t ~ t ~a
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